The Structure and Function of Ribonuclease T₁

XII. Further Studies on Rose Bengal-catalyzed Photooxidation of Ribonuclease T₁-Identification of a Critical Histidine Residue

Abstract

1. The pH dependence of the rate of inactivation of ribonuclease T₁ [EC 2. 7. 7. 26] by rose bengal-catalyzed photooxidation was investigated over the pH range from 5.4 to 9.3. The results were consistent with those of the previous experiments, in-dicating much more clearly the implication of some histidine residue(s) in the in-activation, and hence in the active center of the enzyme. The pH profile indicated the presence of at least one critical histidine residue with an apparent pK value of about 7.5.
2. The enzyme was more or less markedly protected from the inactivation by the presence of one of various substrate analogues. The order of extent of protection was 2'(3')-guanylic acid>5'-guanylic acid>2'(3')-adenylic acid. 2'(3')-Cytidylic acid showed no protective effect under the condition employed. This is consistent with the known substrate specificity of the enzyme as well as with the order of binding ability of the enzyme toward these nucleotides, showing the implication of the active center of the enzyme in the photo-inactivation reaction.
3. Binding ability of the enzyme toward 3' guanylic acid was also greatly decreased by photooxidation, indicating the destruction of the binding site of the enzyme as well.
4. At pH 8.5, histidine-92 was photooxidized most rapidly among the three histi-dine residues in the enzyme and its loss was almost in parallel with the loss of the enzymatic activity. Histidines-27 and -40 were lost much more slowly. Histidine-92 thus appears to be involved in the active center of the enzyme, possibly as part of the catalytic site. In addition, another histidine residue was also suggested to be implicated in the active center, possibly as part of the binding site.